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bioRxiv · 10.64898/2025.12.17.694999

Crosstalk between and developmental dynamics of C. elegans Argonaute proteins

Ketting, R. F.·Seistrup, A.-S.·Nischwitz, E.·Butter, F.
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Abstract

Gene regulation via Argonaute-bound small RNAs is a broadly conserved mechanism, present in all domains of life. The nematode Caenorhabditis elegans expresses several worm-specific Argonautes (WAGOs), which interact with the small RNAs known as 22G-RNAs. These WAGOs have roles in gene regulation, transposon-defence as well as in viral control and experimentally induced RNA interference. Despite many studies, direct relationships between WAGO targeting, as defined by 22G RNA sequences, and mRNA abundance are not clear. Also, the effects of developmental stage and WAGO-interconnectivity have been under-studied thus far. We studied these aspects for two germline-expressed WAGO proteins, WAGO-1 and WAGO-3. We show that WAGO-1 mostly affects 22G-RNA expression in gravid adult worms, while WAGO-3 predominantly affects 22G-RNA expression in embryos. Furthermore, we detect a link between WAGO-3 and the maternal 26G-RNA pathway governed by the Argonaute protein ERGO-1, and between WAGO-1 and the paternal 26G-RNA pathway governed by the Argonautes ALG-3/4. We also demonstrate that, globally speaking, loss of WAGO-1 or WAGO-3 does not result in upregulation of their target mRNAs, as defined by 22G-RNA complementarity. Finally, metagene analysis of 22G-RNA profiles suggests loss of one WAGO protein leads to shifts in WAGO 22G-RNA binding. Overall, we conclude that WAGO-1 and WAGO-3 are developmentally dynamic, are embedded in distinct regulatory networks, and that potential silencing of individual mRNAs by these WAGO proteins is hard to assess by simple loss-of-function studies. SummaryRNA interference describes silencing of genes via small RNAs coupled to an Argonaute protein. C. elegans expresses a class of secondary small RNAs known as 22G-RNAs which are coupled to Argonautes known as WAGOs (worm-specific Argonautes). We have sequenced 22G-RNA and mRNA populations from deletion mutants of two such WAGOs, WAGO-1 and WAGO-3, to investigate Argonaute crosstalk and the relationship between 22G-RNA and mRNA levels. We found that WAGO-1 and WAGO-3 engage different, complementary pathways, and that target silencing cannot be deduced simply from 22G-RNA complementarity.

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Ketting, R. F., Seistrup, A.-S., Nischwitz, E., Butter, F.. 2025-12-19. Crosstalk between and developmental dynamics of C. elegans Argonaute proteins. https://doi.org/10.64898/2025.12.17.694999

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